RESUMO
Integration is a central event in the replication of retroviruses, yet ≥ 90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4(+) T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4(+) T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4(+) T cells. We found that infection of cytokine-treated resting CD4(+) T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Transcrição Reversa , Integração Viral , Replicação Viral , Linfócitos T CD4-Positivos/virologia , DNA Viral/genética , Regulação Viral da Expressão Gênica , HIV-1/genética , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: The integration of HIV-1 DNA into cellular chromatin is required for high levels of viral gene expression and for the production of new virions. However, the majority of HIV-1 DNA remains unintegrated and is generally considered a replicative dead-end. A limited amount of early gene expression from unintegrated DNA has been reported, but viral replication does not proceed further in cells which contain only unintegrated DNA. Multiple infection of cells is common, and cells that are productively infected with an integrated provirus frequently also contain unintegrated HIV-1 DNA. Here we examine the influence of an integrated provirus on unintegrated HIV-1 DNA (uDNA). RESULTS: We employed reporter viruses and quantitative real time PCR to examine gene expression and virus replication during coinfection with integrating and non-integrating HIV-1. Most cells which contained only uDNA displayed no detected expression from fluorescent reporter genes inserted into early (Rev-independent) and late (Rev-dependent) locations in the HIV-1 genome. Coinfection with an integrated provirus resulted in a several fold increase in the number of cells displaying uDNA early gene expression and efficiently drove uDNA into late gene expression. We found that coinfection generates virions which package and deliver uDNA-derived genomes into cells; in this way uDNA completes its replication cycle by viral complementation. uDNA-derived genomes undergo recombination with the integrated provirus-derived genomes during second round infection. CONCLUSION: This novel mode of retroviral replication allows survival of viruses which would otherwise be lost because of a failure to integrate, amplifies the effective amount of cellular coinfection, increases the replicating HIV-1 gene pool, and enhances the opportunity for diversification through errors of polymerization and recombination.
Assuntos
DNA Viral/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Integração Viral/genética , Replicação Viral/fisiologia , Linhagem Celular , DNA Viral/biossíntese , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Infecções por HIV/genética , Provírus/genéticaRESUMO
We hypothesized that estradiol treatment would improve vascular dysfunction commonly associated with obesity, hyperlipidemia, and insulin resistance. A sham operation or 17beta-estradiol pellet implantation was performed in male lean and obese Zucker rats. Maximal vasoconstriction (VC) to phenylephrine (PE) and potassium chloride was exaggerated in control obese rats compared with lean rats, but estradiol significantly attenuated VC in the obese rats. Estradiol reduced the PE EC50 in all groups. This effect was cyclooxygenase independent, because preincubation with indomethacin reduced VC response to PE similarly in a subset of control and estrogen-treated lean rats. Endothelium-independent vasodilation (VD) to sodium nitroprusside was similar among groups, but endothelium-dependent VD to ACh was significantly impaired in obese compared with lean rats. Estradiol improved VD in lean and obese rats by decreasing EC50 but impaired function by decreasing maximal VD. The shift in EC50 corresponded to an upregulation in nitric oxide synthase III protein expression in the aorta of the estrogen-treated obese rats. In summary, estrogen treatment improves vascular function in male insulin-resistant, obese rats, partially via an upregulation of nitric oxide synthase III protein expression. These effects are counteracted by adverse factors, such as hyperlipidemia and, potentially, a release of an endothelium-derived contractile agent.
Assuntos
Estradiol/farmacologia , Resistência à Insulina/fisiologia , Obesidade , Sistema Vasomotor/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Peso Corporal , Relação Dose-Resposta a Droga , Implantes de Medicamento , Ingestão de Alimentos , Inibidores Enzimáticos/farmacologia , Estradiol/sangue , Técnicas In Vitro , Masculino , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Obesidade/fisiopatologia , Ratos , Ratos Zucker , Magreza/fisiopatologia , Vasoconstritores/farmacologia , Vasodilatadores/farmacologia , Sistema Vasomotor/fisiologiaRESUMO
While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.
Assuntos
Adenosina Desaminase/metabolismo , HIV-1/fisiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Adenosina Desaminase/genética , Substituição de Aminoácidos , Líquido da Lavagem Broncoalveolar , Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/classificação , Humanos , Interferon gama/administração & dosagem , Interferon gama/uso terapêutico , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo , Tropismo ViralRESUMO
PURPOSE: Although hypoxia is a known prognostic factor, its effect will be modified by the rate of reoxygenation and the extent to which the cells are acutely hypoxic. We tested the ability of exogenous and endogenous markers to detect reoxygenation in a xenograft model. Our technique might be applicable to stored patient samples. METHODS AND MATERIALS: The human colorectal carcinoma line, HT29, was grown in nude mice. Changes in tumor hypoxia were examined by injection of pimonidazole, followed 24 hours later by EF5. Cryosections were stained for these markers and for carbonic anhydrase IX (CAIX) and hypoxia-inducible factor 1alpha (HIF1alpha). Tumor hypoxia was artificially manipulated by carbogen exposure. RESULTS: In unstressed tumors, all four markers showed very similar spatial distributions. After carbogen treatment, pimonidazole and EF5 could detect decreased hypoxia. HIF1alpha staining was also decreased relative to CAIX, although the effect was less pronounced than for EF5. Control tumors displayed small regions that had undergone spontaneous changes in tumor hypoxia, as judged by pimonidazole relative to EF5; most of these changes were reflected by CAIX and HIF1alpha. CONCLUSION: HIF1alpha can be compared with either CAIX or a previously administered nitroimidazole to provide an estimate of reoxygenation.
Assuntos
Antígenos de Neoplasias/metabolismo , Anidrases Carbônicas/metabolismo , Hipóxia Celular/fisiologia , Etanidazol/análogos & derivados , Hidrocarbonetos Fluorados/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Nitroimidazóis/metabolismo , Oxigênio/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Dióxido de Carbono/farmacologia , Anidrase Carbônica IX , Hipóxia Celular/efeitos dos fármacos , Etanidazol/metabolismo , Feminino , Células HT29 , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Oxigênio/farmacologia , Radiossensibilizantes/farmacologiaRESUMO
Marine and freshwater mussels are notorious foulers of natural and manmade surfaces, secreting specialized protein adhesives for rapid and durable attachment to wet substrates. Given the strong and water-resistant nature of mussel adhesive proteins, significant potential exists for mimicking their adhesive characteristics in bioinspired synthetic polymer materials. An important component of these proteins is L-3,4-dihydroxylphenylalanine (DOPA), an amino acid believed to contribute to mussel glue solidification through oxidation and crosslinking reactions. Synthetic polymers containing DOPA residues have previously been shown to crosslink into hydrogels upon the introduction of oxidizing reagents. Here we introduce a strategy for stimuli responsive gel formation of mussel adhesive protein mimetic polymers. Lipid vesicles with a bilayer melting transition of 37 degrees C were designed from a mixture of dipalmitoyl and dimyristoyl phosphatidylcholines and exploited for the release of a sequestered oxidizing reagent upon heating from ambient to physiologic temperature. Colorimetric studies indicated that sodium-periodate-loaded liposomes released their cargo at the phase transition temperature, and when used in conjunction with a DOPA-functionalized poly(ethylene glycol) polymer gave rise to rapid solidification of a crosslinked polymer hydrogel. The tissue adhesive properties of this biomimetic system were determined by in situ thermal gelation of liposome/polymer hydrogel between two porcine dermal tissue surfaces. Bond strength measurements showed that the bond formed by the adhesive hydrogel (mean = 35.1 kPa, SD = 12.5 kPa, n = 11) was several times stronger than a fibrin glue control tested under the same conditions. The results suggest a possible use of this biomimetic strategy for repair of soft tissues.